Post-translational modifications (PTMs) can significantly modulate the structure and function of proteins and are known to regulate a wide variety of cellular pathways. Dysregulation of PTM pathways have been shown to lead to diverse human diseases, such as cancer, diabetics, and neurodegenerative diseases. PTMs in histones, such as lysine acetylation (Kac) and lysine methylation (Kme), are major epigenetic marks, contributing to the epigenetic program that dictates diverse DNA-templated biological outputs and diseases. Enzymes that regulate histone PTMs other than phosphorylation (such as Kac, Kme, and lysine ubiquitination) have emerged as important drug targets for diseases. We recently reported the identification of a new, evolutionarily-conserved lysine modification, lysine crotonylation (Kcr). We showed that Kcr is abundant in core histones and identified 28 histone Kcr sites in mammalian cells. In male germinal cells immediately following meiosis, histone Kcr, but not histone Kac, is enriched on sex chromosomes and specifically marks testis-specific genes. The unique structure and genomic localization of histone Kcr suggest that it is dynamic and functionally different from histone Kac, a previously-described PTM with diverse functions. Despite of these preliminary studies, major players in the Kcr pathway remain unknown. We hypothesize that Kcr pathway has a unique set of regulatory enzymes, substrates and direct binders, which determines its difference from Kac. We therefore propose to use enzymology, chemical biology, and proteomics approaches to characterize Kcr pathway. Our team is well positioned to carry out this project because of our expertise and the tremendous relevant studies we have carried out in the past few years. In this project, we will first identify and characterize Kcr-regulatory enzymes, crotonyltransferase and decrotonylases, using a variety of chemical biology and enzymology approaches. We will then identify substrates of Kcr and study their dynamics in response to Kcr- regulatory enzymes and during spermatogenesis, by a quantitative proteomics approach. Finally, we will identify and confirm the direct binders for histone Kcr peptides. The new enzymes and binders for histone Kcr are likely to open entirely unforeseen fruitful avenues for histone biology and their roles in disease. Thus, the proposed study should accelerate current research in chromatin biology by revealing novel epigenetic mechanisms, perhaps analogous to the discovery of Kac- and Kme-regulatory enzymes for histone lysine acetylation and histone lysine methylation.